Project 3: Animal Models of FSHD for Therapy Development FSHD is one of the more common forms of muscular dystrophy yet the underlying cause(s) of the clinical symptoms seen in patients are still unclear. During the first period of support of our Wellstone Center, critical discoveries have been made by us and others on some of the key molecular players in disease causation. The open reading frame in the last D4Z4 repeat encoding the protein termed DUX4-FL clearly plays a role in disease progression. The misexpression of DUX4-fl and it's downstream target transcripts likely play a role in the disease but which are important for pathogenesis are still unclear. There is a large unmet need in available animal models of FSHD for the study of pathogenesis and the development of novel therapeutics. Project 3 of The Wellstone Center renewal aims to use xenografts of mouse and human muscle as well as a zebrafish model of DUX4 misexpression during development. The humanized mouse muscle either by direct engraftment of human skeletal muscle or with cell transplantation provides human muscle in a living organism on which to develop therapeutic approaches. The models should allow the search for modulators of DUX4-fl expression and any key developmental targets of DUX4-fl expression. In addition, the latter should result in an understanding of the generation of clinical symptoms outside skeletal muscle. Knock down of Dux4-fl through AAV as well as morpholino administration will be developed in the xenografts with the ultimate goal of using these approaches in clinical trials. The project will be informed by Project 1 and Project 2 on additional candidate genes and biomarkers whose expression will be assayed in the models. The Project will also rely heavily on the Cell Core for well characterized FSHD and unaffected control cells and muscle biopsies.